Chromosomal aberrations are a leading cause of genetic disorders or diseases, including congenital disorders and acquired diseases such as malignancies. At the base of these malignancies is the fact that all cells of a malignancy have a common clonal origin. Chromosomal aberrations in malignancies stem from rearrangements, translocations, inversions, insertions, deletions and other mutations of chromosomes, but also losses or gains of whole chromosomes are found in malignancies. In many chromosome aberrations, two different chromosomes are involved. In this way, genes (or fragments of genes) are removed from the normal physiological context of a particular chromosome and are located to a recipient chromosome, adjacent to non-related genes or fragments of genes (often oncogenes or proto-oncogenes). Such an aberrant genetic combination can be the foundation of a malignancy.
Often, such rearrangements involving two non-aberrant chromosomes happen in a somewhat established pattern. Breaks occur in either of the two chromosomes at a potential breakpoint or breakpoint cluster region, resulting in the removal of a gene or gene fragment from one chromosome and subsequent translocation of the gene or gene fragment to the other chromosome, thereby forming a rearranged chromosome where the rearranged fragments are fused in a fusion region.
Detection of chromosome aberrations can be achieved using a wide array of techniques, various of which entail modern biomolecular technology. Traditional techniques such as cytogenetic analyses by conventional chromosome banding techniques are, although highly precise, very labor intensive, require skilled personal and are expensive. Automated karyotyping is useful for some diagnostic applications, such as prenatal diagnosis, but is ineffective in analyzing the complex chromosomal aberrations of many malignancies. Furthermore, the above techniques require fresh (cultured) cells, which are not always available.
Other, more modern, techniques are Southern blotting or other nucleic acid hybridization techniques or amplification techniques such as polymerase chain reaction (“PCR”) for the detection of well-defined chromosome aberrations for which suitable nucleic acid probes or primers are available. With these techniques, fresh or frozen cells and sometimes even samples after formalin fixation can be used, as long as the nucleic acid sequences to be hybridized or amplified remain intact and accessible. However, even with this modern technology, several disadvantages can be found that hamper the application of these diagnostic techniques in the rapid screening for chromosomal aberrations related to such malignancies can be found.
Southern blotting lasts 3 to 4 weeks, which is too slow for efficient diagnosis and choice of therapy in malignancies, and allows only 10–15 kb of nucleic acid sequences to be analyzed per probe analysis.
PCR, although, in essence, well-suited for rapid and massive diagnostic testing or even screening, allows only 0.1 to 2 kb of nucleic acid to be analyzed per PCR analysis, which greatly hampers rapid screening of vast stretches of chromosomes and breakpoint cluster regions within the chromosomes. An additional disadvantage of PCR is its inherent sensibility to mismatched primers. Small, normal, and physiological alterations which can always be present in the nucleic acid sequence of the gene fragment complementary to the primer hamper the reliable application of PCR and eventually give rise to false-negative results, which renders a PCR-based diagnostic test, albeit very specific, not sensitive enough for reliable diagnosis. Only a reliable diagnosis of malignancies can contribute to an understanding of the prognosis and the design of an adequate therapy.
Fluorescent in situ hybridization (“FISH”) techniques are less dependent on the complete matching of nucleic acid sequences to provide positive diagnostic results. In general, FISH employs probe analyses with large, mainly unspecified, nucleic acid probes that hybridized, however, often with varying stringency, with the genes or gene fragments located at both sides of the fusion region in the rearranged chromosome in the malignant cell. Using large probes renders the FISH technique very sensitive. The binding of the co-localizing probes is generally detected either directly or indirectly with fluorochromes and visualized via fluorescence microscopy of a population of cells obtained from the sample to be tested.
However, even the currently used FISH protocols have inherent disadvantages. These disadvantages mainly relate to the selection of nucleic acid probes employed in the current FISH protocols, which can give false-positive results in the diagnosis of chromosomal aberrations. For example, probes directed against different chromosomes with a juxtaposition of signals in the case of translocation create a rather large risk of false-positive results. Hence, the diagnostic tests, although sensitive, are not specific enough to employ standard FISH techniques in massive or rapid diagnostic testing, let alone in automated testing or screening.
Thus far, generally large probes derived from cosmic clones, YAC clones, or other cloned DNA fragments, have been used as probes in FISH. The exact position of these probes in relation to the fusion region in the rearranged chromosome is unknown and these probes are of largely unspecified and varying genomic length (genomic length or distance as expressed as the number of nucleotides or bases (b)) and go, without specific selection or modification of these probes, beyond the mere labeling of the probes with the necessary reporter molecules, i.e., fluorochromes. For designing or selecting probes, little or no guidance is given in the art beyond mere suggestions as to where to localize a putative probe. False-positive results obtained with these probes may stem from a specific hybridization with a wide array of (major) repetitive sequences present throughout various chromosomes, or from cross-hybridization to homologous sequences in the genome, or from overlap of the probes used with the breakpoint cluster region or from the difference in signal intensities as far as originating from size differences of the probes. These causes of false-positive results are frequently not recognized. False-positive results are especially detrimental to rapid diagnosis if rapid or routine screening of patients is needed to detect malignancies or in evaluating treatment protocols. A false-positive result then necessitates cumbersome retesting of patients, or even unsuspecting clients that have been submitted to routine screening protocols, and can greatly alarm these people. Furthermore, translocations are generally detected with two different probes, one for each of the involved chromosomes, which probes then colocalize during the in situ hybridization in the case of a translocation, but show separate signals when no translocation is present (see, e.g., European patent applications EP 0430402 and EP 0500290; Tkachuk et al., Science 250:559–562 (1990); Tkachuk et al., “Clinical applications of fluorescence in situ hybridization,” Genetic analysis techniques and applications 8:67–74 (1991). However, in practice, 2 to 4% of normal interphase cells tested by FISH will show false-positive results due to the fact that the two probes colocalize by chance. An additional disadvantage of the current FISH protocols is that it is, in practice, necessary to know both chromosomes that are involved in the translocation as well as the relevant breakpoint regions of both chromosomes to define the nucleic acid probes enabling the detection of the specified translocation, while as yet unknown or ill-defined translocations originating from a well-known gene and an unknown partner gene remain undetected.